One gene, two phenotypes – understanding the pathomechanics and leukemia development in congenital neutropenia and cyclic neutropenia

重症先天性中性粒细胞减少症（CN）是一种单系白血病前骨髓衰竭综合征，其特征是早期发作的中性粒细胞减少症伴严重感染。CN是一种异质性疾病，由不同基因突变引起，但最常见于ELANE，编码中性粒细胞弹性蛋白酶（NE）蛋白，其作为常染色体显性遗传疾病遗传。遗传性中性粒细胞减少症的第二种形式是周期性中性粒细胞减少症（CyN），其特征在于中性粒细胞计数的振荡，每21天出现一个最低点。在低中性粒细胞计数期间，患者可能遭受感染，如口疮性口炎、牙周炎和盲肠炎。几乎所有的CyN病例都是由ELANE突变引起的，值得注意的是，引起CyN的突变总是与CN中描述的突变重叠。目前还不清楚相同的突变如何导致CN和CyN。CN的一个严重并发症，以及CyN的一个小得多的程度，是骨髓增生异常综合征（MDS）和急性髓性白血病（AML）的发展。粒细胞集落刺激因子（G-CSF）是CN和CyN患者的救命药物，可改善粒细胞生成并预防严重感染。然而，CN中发生白血病的风险与所需的G-CSF剂量直接相关。在G-CSF治疗10年后，CN中MDS或AML的累积发生率约为20%。CyN患者发生MDS和AML的易感性要低得多，但仍然存在。目前尚不清楚为什么CyN患者的白血病进展要低得多。不存在研究由ELANE突变引起的CN和CyN的小鼠模型，因为在小鼠中靶向ELANE的小鼠直系同源物不会引起中性粒细胞减少症。此外，由于CN和CyN通常是由ELANE中相同的突变引起的，因此不可能通过简单的基因编辑方法生成区分这两种疾病的准确模型。因此，我们的目标是使用患者来源的诱导多能干细胞（iPSC）生成ELANE突变引起的CN和CyN以及CN恶性转化的模型。重要的是，iPSC携带患者的完整遗传背景，因此将包括迄今为止尚未确定的任何可能的遗传修饰剂。我们最近发表了患者来源的iPSC的生成和粒细胞分化，包括ELANE突变引起的CN。iPSC概括了临床上观察到的有缺陷的粒细胞生成。没有发表的研究利用CyN的iPSC模型。我们计划使用患者来源的iPSC比较CN和CyN的病理机制。此外，由于CN患者携带与获得性G-CSFR突变相关的白血病转化的高风险，我们的目标是通过使用CRISPR/Cas9引入这些突变来生成白血病前期的模型。我们相信，这一模型将有助于阐明参与CN的早期恶性转化过程的途径，并可能揭示更一般的途径，有助于恶性转化。

Severe congenital neutropenia (CN) is a mono-lineage pre-leukemia bone marrow failure syndrome, characterized by early onset of neutropenia accompanied by severe infections. CN is a heterogeneous disease, caused by mutations in different genes, but most commonly in ELANE, encoding the neutrophil elastase (NE) protein, which is inherited as an autosomal dominant disease. A second form of hereditary neutropenia is cyclic neutropenia (CyN), which is characterized by oscillations of the neutrophil counts with a nadir occurring every 21 days. During the periods of low neutrophil counts, patients may suffer from infections, such as aphthous stomatitis, periodontitis and typhlitis. Almost all cases of CyN result from mutations in ELANE and, remarkably, the mutations causing CyN invariably overlap with those described in CN. It is unclear how the same mutation can cause CN and CyN. One severe complication of CN, and to a much lesser extent of CyN, is the development of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Granulocyte-colony stimulating factor (G-CSF) is a life-saving drug for CN and CyN patients, improving the granulopoiesis and preventing severe infections. However, the risk of leukemia development in CN directly correlates with the G-CSF dose required. The cumulative incidence of MDS or AML in CN is around 20% after 10 years of G-CSF treatment. Patients with CyN have a much lower but existent susceptibility to develop MDS and AML. It is unclear why leukemia progression is much lower in CyN patients. No mouse model exists to study CN and CyN caused by ELANE mutations, as targeting the mouse ortholog of ELANE in mice does not cause neutropenia. In addition, as CN and CyN are often caused by identical mutations in ELANE, it is not possible to generate accurate models that will differentiate between the two disorders by simple gene editing methods. Therefore, we aim to generate a model for CN and CyN caused by ELANE mutations and for the malignant transformation in CN, using patients-derived induced pluripotent stem cells (iPSCs). Importantly, iPSCs carry the full genetic background of the patients, and will therefore include any possible genetic modifiers that have not been identified so far. We recently published the generation and granulocytic differentiation of patients-derived iPSCs, including for CN caused by mutations in ELANE. iPSCs recapitulated the defective granulopoiesis observed clinically. No published study utilized an iPSCs model of CyN. We plan to compare pathomechanics of CN and CyN using patients-derived iPSCs. In addition, as CN patients carry a high risk for leukemic transformation, related to acquired G-CSFR mutations, we aim to generate a model for pre-leukemia by introducing these mutations using CRISPR/Cas9. We believe that this model will help elucidate the pathways involved in early malignant transformation processes in CN and may reveal more general pathways contributing to malignant transformation.